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A radioimmunoassay for human insulin receptor correlation between insulin binding and receptor mass
1Department of Medicine, Temple University School of Medicine, Philadelphia, Pennsylvania.
Insights
Researchers developed a sensitive radioimmunoassay to measure human insulin receptor mass. This assay indicates that one insulin molecule binds per human placental insulin receptor, aiding in understanding insulin resistance.
Area of Science:
- Endocrinology
- Immunology
- Biochemistry
Background:
- Severe insulin resistance is a complex metabolic disorder.
- Accurate quantification of insulin receptor (IR) is crucial for understanding its role in insulin resistance.
- Existing methods may not precisely measure IR mass independently of its binding activity.
Purpose of the Study:
- To develop and validate a sensitive radioimmunoassay (RIA) for quantifying human insulin receptor (hIR) mass.
- To determine the molar ratio of insulin binding sites to hIR mass in human placenta.
- To investigate the stoichiometry of insulin binding to the hIR.
Main Methods:
- Development of a RIA using anti-hIR antiserum from a patient with Type B insulin resistance.
- Utilization of purified human placental IR as a reference and 125I-labeled purified hIR as a tracer.
- Measurement of insulin binding using a standard insulin binding assay and IR mass via the developed RIA in placental tissue.
Main Results:
- The developed RIA demonstrated high sensitivity (limit of detection < 17 fmol), reproducibility (CVs 12.5% intra-assay, 1.6% inter-assay), and specificity.
- The assay distinguished between human placental and rat liver IR and showed no cross-reactivity with IGF-1 receptor, insulin, or glucagon.
- The molar ratio of insulin binding to IR mass was approximately 0.86:1 with monoiodinated insulin and 1.94:1 with randomly iodinated insulin.
Conclusions:
- A novel, sensitive, reproducible, and specific RIA for human insulin receptor mass has been established.
- The RIA allows for the measurement of IR mass independently of insulin binding capacity.
- Data suggest a stoichiometry of one insulin molecule binding per human placental insulin receptor.
Abstract:
We have developed a radioimmunoassay for human insulin receptor. Serum from a patient with Type B severe insulin resistance was used as anti-insulin receptor antiserum. Pure human placental insulin receptor was used as reference preparation and 125I labeled pure insulin receptor as trace. The radioimmunoassay was sensitive (limit of detection less than 17 fmol), reproducible (inter and intra-assay coefficients of variation 12.5% and 1.6% respectively) and specific (no crossreactivity with pure placental IGF-1 receptor, insulin and glucagon). The anti-insulin receptor antibody was, however, able to differentiate between insulin receptor from human placenta and from rat liver. To determine the number of insulin binding sites per receptor, we measured insulin binding (by insulin binding assay) and insulin receptor mass (by radioimmunoassay) in solubilized aliquots from 5 human placentas. The molar ratio of insulin binding to receptor mass was 0.86 +/- 0.12 when binding was determined with monoiodinated 125I-Tyr A 14-insulin. It was 1.94 +/- 0.27 when randomly iodinated 125I-insulin was used. In conclusion, using a sensitive, reproducible and specific radioimmunoassay, we have measured insulin receptor mass independent of insulin binding. Our data are most compatible with binding of one insulin molecule per human placental insulin receptor.
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